Project Summary Minimal improvement in the 12-15 month average survival of patients with glioblastoma multiforme (GBM) has been achieved despite decades of advances in neurosurgery and radiation therapy, many clinical trials for novel therapeutics, and increased understanding of the driving molecular mechanisms. A central issue that confounds successful treatment is the heterogeneous nature of this aggressive tumor. This heterogeneity presents phenotypically as mixed cytological subtypes, genotypically as mutations and gene amplifications, and transcriptionally as regional differences in gene expression. As a result, multiple and spatially distinct heterotypic populations exist within a single GBM, making any lesion- or pathway-specific therapy less effective. While considerable effort has been placed on understanding cell intrinsic mechanisms conferring therapeutic resistance, much less is known about the interactions between heterogeneous tumor cells within these neoplasms that contribute to the recalcitrant nature of this cancer. In GBM, amplification of the epidermal growth factor receptor, a hallmark mutation present in 60% of cases, often occurs in a heterogeneous manner and is frequently associated with structural alterations. The most common of these alterations, EGFRvIII, (also known as ?EGFR) results in a constitutively active mutant receptor with tumor enhancing capability. This ability is lacking from amplified wtEGFR despite its more pervasive tumor expression. By modeling this type of genetic heterogeneity in vivo, we have determined that an IL-6 paracrine signaling mechanism driven by EGFRvIII activity can not only recruit wtEGFR-expressing cells into accelerated proliferation, but also promote EGFR-targeted therapeutic resistance through activation of a pro-survival inflammatory NF-?B/BRD4 signaling axis. Given the central role of NF-?B/BRD4 in the remodeling of chromatin super enhancers we postulate that EGFRvIII/wtEGFR sub-population interactions not only enhance aggressive tumor growth, but also prompt the synchronization of aspects of gene expression in these heterotypic cells through shared enhancer remodeling. The overall goal of this renewal project is to dissect and target the mechanisms whereby GBM EGFR/EGFRvIII heterogeneity drives therapeutic resistance through orchestration of NF-?B/BRD4-mediated remodeling of the epigenetic landscape. The following lines of experimentation will be carried out: 1) genome editing to create a drug-selective BRD4 allele for mechanistic chemical biology studies; 2) use of this modified BRD4 allele as a tool for chromatin structure and functional analysis of the cytokine-stimulated NF-?B/BRD4 enhancer landscape in heterotypic and subpopulation-ablated gliomas; 3) genetic and pharmacological inhibition of identified NF-?B/BRD4-mediated genetic or epigenetic vulnerabilities shared among EGFR and EGFRvIII heterotypic cells to enhance therapeutic efficacy.